Preparative electrophoretic separation of proteins and other compounds, i.e. separation by charge within an electric field, is normally carried out in a column of anticonvectant medium, e.g. chemical gel. The samples are delivered to the upper surface of the gel, electric potential is applied and, after a time interval, separated components emerge at the bottom of the gel column. The scale of this method of preparative separation is limited by the cross section of the gel which must not exceed the capacity of the system to remove heat generated by the current. This cross section can only be of the order of several square millimeters because of the very poor heat conductivity of the gel. The present invention aims to overcome these limitations in a number of ways.
It is also known to carry out electrophoretic separation as between free flowing streams either with free or fixed boundaries. In free boundary electrophoretic separation macromolecular species in solution, including colloidal solution, are separated as a stream of the solution is passed between charged electrodes. The stream is then divided, without remixing, into a plurality of parts with differing proportions of the molecular species present in the original stream (see U.S. Pat. No. 2,878,178, and British Patent specification 1,255,418). In fixed boundary electrophoretic separation a semi-permeable membrane acts as a filter through which the liquid stream passes (see U.S. Pat. No. 3,079,318) or acts to separate two streams of liquid between which at least one molecular species migrates under the electrophoretic influence (see PCT Patent specification W079/00002 and U.S. Pat. No. 3,989,613).
A problem with fixed boundary electrophoresis is the fouling or clogging of the membranes. The efficiency of the process depends upon:
1) maximum contact of the processed solution with the membrane surface, and PA1 2) unimpaired permeability of the membrane pores intended to permit the passage of the macromolecules in question. PA1 1) Upstream fluid phase; PA1 2) Upstream interface with membrane; PA1 3) Membrane gel; PA1 4) Downstream interface; PA1 5) Downstream fluid phase; PA1 1. Electrophoretic migration in the upstream fluid will be disturbed by a rapid turbulent flow. On the other hand a flow rate may be so slow that all the relevant components will migrate to the proximal part of the membrane, and a large distal part of the membrane may not participate in the process. Thus a preferred flow pattern in this phase is stepwise with one step following: designed to replace quickly the spent solution with a fresh one, followed by a stationary period to allow migration to the membrane. The optimal volume and duration of each cycle will depend on the prevailing conditions. PA1 2. The upstream fluid/membrane boundary may be blocked by obstructed molecules forming an insoluble film on the surface, a condition called "fouling" in filtration processes. When necessary this can be counteracted by temporarily reversing the electric field toward the end of each cycle to detach the aggregating molecules. Frequency and duration of field reversal must be determined experimentally and may be controlled manually or automatically by monitoring changes in electrical conductivity of the stack during each phase or cycle. PA1 3. Within the membrane the transport is dependent mainly on the characteristics of current and membrane composition and is isolated from the flow mechanics. PA1 4. In the downstream fluid phase a vigorous cross flow is desirable because it prevents molecules adhering to limiting membranes separating the solvent from the buffer streams and offers the opportunity for heat removal with the aid of an external heat exchanger. PA1 1. Stroke 1 (e.g. 5 seconds). Plunger out, pump filled with the solution withdrawn from the stack (replaced by fresh fluid sucked from the reservoir). PA1 2. Interval (e.g. 60 seconds) for electrophoretic transport, while the plunger is being slowly depressed, expelling pump effluent to a collection or recirculation reservoir. Current may be reversed by a linked mechanism during the last few seconds of the cycle.
Maximum contact, in turn, depends on uniform distribution of fluid flow in the spaces between the membranes. Because of the relatively slow passage of the fluid in electrophoretic separation manifolding of the flow through parallel spaces in a stack of electrophoretic membranes as well as ensuring a uniform flow of the fluid film along each space may be difficult because of preferential channelling due to slight differences in the geometry, air locks and similar imperfections. The present invention aims, in a first aspect, to overcome this problem.
It will be realised that membrane fouling or clogging can occur in fixed boundary electrophoretic separation when the pores in the membranes approach in size the molecules being separated. In a second aspect the present invention aims to overcome this problem.
A further problem with known fixed boundary electrophoretic separation has been the difficulty in maintaining a free flow of solvent between adjacent membranes. In a third aspect the present invention provides means to effectively space adjacent membranes while allowing free flow of solute therebetween.
A still further problem with known fixed boundary electrophoretic separation apparatus has been the physical problems of filling, emptying, cleaning and reassembling of a housing containing the electrodes and membranes. Conventionally such a housing has been formed substantially integrally with associated storage tanks, pumps, cooling apparatus and the like making the housing itself difficult to handle. In a fourth aspect the present invention provides improved apparatus for overcoming these physical handling problems.